Smooth muscle cell proliferation is the hallmark of many diseases, including pulmonary hypertension, asthma, lymphangioleiomyomatosis, and tuberous sclerosis complex (TSC). Hamartin and tuberin are highly expressed in smooth muscle and are encoded by the tumor suppressor genes, Tuberous Sclerosis Complex-1 and -2 respectively (TSC1; TSC2). Tumor suppressor function is dependent upon complex formation between hamartin and tuberin. Functional loss of either hamartin or tuberin leads to identical cellular effects: Increased levels of the G protein, GTP-Rheb (Ras homolog enriched in brain), with downstream constitutive activation of mTOR (mammalian target of rapamycin), S6Kinase (S6K) and S6, leading to increased cellular growth. We show that independent of S6K-S6 activation, the cellular hyperproliferation observed in pulmonary artery vascular smooth muscle cells (PaVSMCs), where the TSC1/TSC2 complex is disrupted, is also associated with (i) rapamycin-sensitive effects that involve mitochondrial-derived superoxide anion (O2.-) production and reduced mitochondrial-mediated apoptosis and (ii) rapamycin-insensitive activation and co-localization of the G proteins, Rheb and Rho A. Recent data suggests that low grade pulmonary hypertension occurs in women diagnosed with pulmonary LAM, a disease where TSC2 is functionally deficient and we have observed increased pulmonary pressures in a murine model where TSC1 is conditionally knocked out in vascular smooth muscle. Our hypothesis is that in PaVSMCs, TSC1/TSC2 complex dysfunction results in: (i) rapamycin- sensitive altered mitochondrial function that promotes redox-regulated apoptosis, (ii) rapamycin- insensitive activation of Rheb and RhoA that also promotes growth, and (iii) a phenotype that promotes the development of pulmonary hypertension. To address this hypothesis , our Specific Aims are: 1) To study the regulation by the TSC1/TSC2 complex of the rapamycin-sensitive generation of mitochondrial O2.- and the downstream effectors that are pertinent to PaVSMC growth; 2) To study the regulation by the TSC1/TSC2 complex of the rapamycin-insensitive activation of the G proteins Rheb and RhoA and their downstream effectors pertinent to PaVSMC growth and 3) To study the effect of TSC1/TSC2 complex disruption on the development of pulmonary hypertension in vivo. These studies will lead to a new understanding of how the TSC1/TSC2 complex controls the proliferation of pulmonary vascular smooth muscle. We hope that our studies will lead to the development of new therapies for diseases characterized by SMC proliferation like pulmonary hypertension. PUBLIC HEALTH RELEVANCE: Smooth muscle cell (SMC) growth is the hallmark of pulmonary hypertension. We plan to study the regulation of SMC growth by two SMC proteins, hamartin and tuberin. The purpose of the proposed studies is to improve our understanding of the mechanisms that lead to uncontrolled growth in smooth muscle and to develop new targets for the treatment of pulmonary hypertension.